The epithelial lining of the gastrointestinal tract forms a vital protective barrier that separates luminal antigens and toxins from the underlying tissue compartments. Patients with inflammatory bowel disease (IBD) encompassing both Crohn's disease and ulcerative colitis present clinically with relapsing intestinal inflammation and diarrhea. Numerous mechanisms have been proposed to explain these clinical symptoms and include defective intestinal epithelial barrier function. Increased paracellular permeability has been documented in the epithelial lining from both the acutely inflamed and chronically damaged areas of the intestine. Epithelial barrier function is regulated to a large extent by the apical most intercellular junction referred to as the tight junction (TJ). The TJ not only separates the lumenal compartment from the tissue space, but has been shown to regulate movement of solutes across the paracellular space in diverse physiologic and pathologic states. The transmembrane proteins in epithelial TJs include occludin, members of the claudin family, junctional adhesion molecule (JAM)-A and coxsackie and adenovirus receptor (CAR). Our studies indicate that JAM-A is a key TJ protein with several functions important in regulating intestinal epithelial barrier, cell shape and cell-matrix adhesion. In this proposal, we continue a structure-function based approach to study human JAM-A. The specific aims of this proposal are focused on determining intracellular events that mediate JAM-A function and the structural basis of extracellular homophilic interactions of JAM-A responsible for this. We will primarily utilize in vitro cell culture systems amenable to dissecting out biochemical and molecular events in intestinal epithelial cell lines in concert with in vivo studies in mice to highlight relevance. The long term goal is to correlate our findings on the molecular regulation of JAM-A function with pathophysiology in IBD. Understanding basic mechanisms regulating intercellular junctions and paracellular movement of fluids and solutes may provide clues to the pathophysiology of mucosal diseases such as IBD and aid in the development of new therapeutic strategies aimed at diminishing enhanced permeability and mucosal injury associated with these conditions.